1. Field of the Invention
The ability to isolate DNA sequences coding for naturally occurring polypeptides and introducing the isolated sequences into foreign hosts for expression of the encoded polypeptide has greatly expanded the opportunities to produce polypeptides or analogs thereof having biological activity. As more is learned about the mechanism of expression in both prokaryotes and eukaryotes, there is an expanding appreciation of the sophistication of the mechanisms of transcription and translation, as well as replication, and the interaction of naturally occurring segments, both coding and non-coding. While various techniques have been developed which aid in the isolation, sequencing and synthesizing of DNA and peptides, each investigation of expression of a new polypeptide frequently results in unanticipated obstacles, which require new approaches or modifications of existing techniques.
It has long been known that B. thuringiensis species produce a variety of proteins having insecticidal activity. Because of their natural nature, biodegradability, and the apparent absence of insect resistance to their toxicity, these insecticides have attracted much interest. However, due to the difficulties of growing the host for producing the insecticidal protein, the use of these naturally-occurring insecticides has been somewhat limited. It has therefore become of interest to develop DNA sequences coding for proteins having the same or analogous properties to the naturally occurring B. thuringiensis insecticides.
The endotoxin is formed in a proform as a crystalline inclusion body. The effectiveness of a soluble form, the possibility of instability to proteolytic or other degradation and the ability to produce active fragments, which may have substantial advantages in economics and activity remain to varying degrees a matter of conjecture.
2. Description of the Prior Art
The Bacillus thuringiensis var. israelensis (BTI) endotoxin has been suggested to be associated with the large plasmids of this species. Kamdar and Joyaraman, Biochem. Biophys. Res. Commun. (1983) 110:477-482; and Clarke et al., (1983) Abstracts of the Annual Meeting of the American Society for Microbiology: H91. Properties of BTI endotoxin are described by Thomas and Ellar, J. Cell Sci. (1983) 60:181-197; Tyrell et al., J. Bacteriol. (1981) 145:1052-1062; and Clarke et al., supra. Properties of the kurstaki endotoxin as produced by recombinant plasmids is described in Schnepf and Whitely, Proc. Natl. Acad. Sci. USA (1981) 78:2893-2897, reporting that a soluble, rather than crystalline form is obtained.